1. Field of the Invention
This invention generally relates to the field of diagnostic virology and, more particularly, to a method for detecting infectious herpes virus in a specimen and a cell line for use therefor.
2. Description of the Related Art
Herpesviruses have become increasingly important causes of human morbidity and mortality. Whitley, R. J., Virology (1990) New York, Raven Press. Herpes simplex viruses Type 1 and 2 (hereinafter referred to collectively as HSV), in particular, infect a large number of individuals each year. Primary infection of immunocompetent patients with HSV usually leads to a mucocutaneous syndrome such as herpes labialis or herpes genitalis, the latter being one of the most common sexually transmitted diseases today. Infection with HSV can also cause more serious infections, the most serious of which are sight-threatening keratitis and life-threatening encephalitis. Moreover, HSV related disease in immunocompromised individuals such as newborns, leukemia patients, organ transplant recipients and AIDS patients has become an increasingly prevalent and difficult problem.
Significant advances have been made in the treatment of HSV infections in the past decade. These advances in antiviral therapy have expanded the role of the diagnostic virology laboratory and have identified the need for more sensitive, accurate and rapid diagnostic tests to assist in the early diagnosis of HSV infections.
Various tests are presently available for the diagnosis of HSV infections. Most involve the detection of viral antigens or intact infectious virus. Antigen detection assays offer the advantage of rapidity and specificity, but can lack the necessary sensitivity. Kowalski, R. P. and Gordon, Y. J., Ophthal. (1989) 96:1583-1586. The most reliable test to detect infectious herpes virus involves inoculation of specimens onto tissue culture cells followed by detection of infectious virus by microscopically observing a characteristic cytopathic effect. Although HSV is a relatively easy virus to culture as it replicates on a wide variety of continuous cell lines, virus propagation in tissue culture can be slow and expensive. Recently, improved techniques have been developed for the detection of viruses from clinical specimens. The shell vial technique, for instance, has greatly increased the sensitivity and the rapidity of HSV detection. When this method is combined with antigen detection by immunohistochemistry, HSV can be positively identified within 24 hours in the majority of cases. Gleaves et al., J. Clin. Micro. (1985) 21:29-32; Ziegler et al., J. Clin. Micro. (1985) 26:2013-2017. While this type of assay is preferred in diagnostic virology applications, it is labor intensive and a significant number of specimens are not identified as positive until after 48 hours. Another recent technological advance, polymerase chain reaction (PCR) technology, presents a promising tool for the detection of HSV particularly in cerebrospinal fluid specimens, but this technology detects viral nucleic acid and not infectious virus. Puchhammer-Stockl, et al., J. Med. Virol. (1990) 32:77-82. The detection of infectious virus is often preferred because it definitively indicates that there is an ongoing viral infection with active viral replication. PCR detection of viral nucleic acid may only be indicative of the presence of a remnant of a past infection or the presence of a latent infection.
Previous scientific studies involving herpes viruses have used susceptible cell lines transfected with a chimeric DNA construct containing a marker gene in transient assays to study various aspects of the virus such as the regulation of gene expression during viral replication. Flanagan W. M. and Wagner, E. K., Virus Genes 1:1:61-71 (1987). These studies have not, however, described a DNA construct stably integrated into the chromosome of a stable cultured cell line which is suitable for the diagnostic detection and quantification of a herpes virus in a specimen with the requisite sensitivity and specificity for a clinical diagnostic assay.
Recently, a method for detecting infectious HIV in a specimen has been disclosed that utilizes a genetically engineered cell line containing a chimeric gene having the E. coli LacZ gene associated with the HIV-1 LTR promoter. Rocancourt, et al., J. Virol. (1990) 64:2660-2668; Kimpton, J. and Emerman, M., J. Virol. (1992) 66:4:2232-2239. Although these cell lines may be useful for detecting HIV in a specimen, they are not suitable for diagnostic virology assays because of their lack of specificity. It is well-known that the HIV-1 LTR promoter used in the DNA construct of these studies to cause expression of the reporter gene is not specific for HIV and that other viruses cause expression of the reporter gene if present in the specimen. In particular, the presence of HSV or cytomegalovirus in the specimen causes activation of the LTR promoter and subsequent expression of the reporter gene even in the absence of HIV in a specimen. Mosca, J. D., et al., Nature (1987) 325:67-70; Mosca, J. D., et al., Proc. Natl. Acad. Sci. (1987) 84:7408-7412; Popik and Pitha, Proc. Natl. Acado Sci. (1981) 88:9572-9577. If such a cell line were used in a diagnostic assay, it could lead to the erroneous diagnosis of the presence of HIV in a specimen when in fact the specimen contained a different virus. Thus, the lack of specificity in cell lines prepared to detect HIV in a specimen prevents their use in a diagnostic assay which requires specificity.
A need exists, therefore, for a method for detecting a herpes virus in a specimen that provides rapid detection in a cost efficient manner, while also providing the sensitivity and specificity necessary for a diagnostic assay.